Project Summary: Kinesin motors carry out bidirectional transport in neurons, organize the mitotic spindle during cell division, and are involved in a number of other vital cellular processes. Significant progress has been made in understanding the mechanochemical transitions that underlie kinesin stepping; however, identifying the key structural features and the rates of structural and biochemical transitions that underlie the functional diversity observed across the kinesin superfamily remains elusive. Our ongoing work suggests that: i) differences in unloaded processivity between kinesin families result from differences in the length of their neck linker domains, a 14-18 residue sequence connecting the catalytic core to the coiled-coil domain, and ii) differences in motors' response to load result from properties resident in the core motor domain. We hypothesize that the processivity of dimeric kinesins results from a race between attachment of the tethered head to the next tubulin binding site and dissociation of the bound motor from the microtubule. Until now, there has been no reliable way to the detachment/reattachment dynamics of one head in a processive kinesin dimer. We will attach gold nanoparticles to individual heads in a kinesin dimer and track the particles with nm and sub-msec resolution using Interferometric Scattering (iSCAT) and Total Internal Reflection Dark-Field (TIRDF) Microscopy. We recently showed that iSCAT can detect substeps at saturating ATP levels that have not been measured by optical tweezers, the benchmark in the field. Aim 1 is to identify transitions in the mechanochemical cycles of kinesin-1, the intraflagellar transport motor kinesin-2, and the `superprocessive' kinesin-3 that govern their differing behaviors. To understand how kinesins transport cargo along the crowded microtubules found in cells, Aim 2 will identify features of the hydrolysis cycle that enable motors to step around roadblocks. In Aim 3 these investigations will expand to the mitotic motor kinesin-5, which we recently found to be a microtubule polymerase. Experiments in Aim 3 will identify the specific features of the kinesin-5 chemomechanical cycle that lead to its plus-end-binding and microtubule polymerase activity. We will test the hypothesis that the contrasting influence of load on these motors results from what proportion of the hydrolysis cycle the motor resides in a vulnerable one-head-bound state. By applying new techniques to measure rapid mechanical transitions during kinesin stepping, this work will help to define a general model for kinesin mechanochemistry that can be applied to diverse motors in the superfamily. This understanding is necessary to extrapolate observations at the single-molecule level to the complex dynamics of multi-motor transport in cells in both normal and diseased states.